1、Designation: D6584 10a1Standard Test Method forDetermination of Total Monoglycerides, Total Diglycerides,Total Triglycerides, and Free and Total Glycerin in B-100Biodiesel Methyl Esters by Gas Chromatography1This standard is issued under the fixed designation D6584; the number immediately following
2、the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1NOTECorrected formatting and spac
3、ing in Eqs 12-19 and added previous Summary of Changes editorially in March 2011.1. Scope*1.1 This test method covers the quantitative determinationof total monoglyceride, total diglyceride, total triglyceride, andfree and total glycerin in B-100 methyl esters by gas chroma-tography. The range of qu
4、antitation for monoglyceride is 0.100to 1.000 mass %, for diglyceride is 0.050 to 0.500 mass %, andfor triglyceride is 0.050 to 0.500 mass %. The range ofquantitation for free glycerin is 0.005 to 0.05 mass % and fortotal glycerin from 0.05 to 0.5 mass %. This procedure is notapplicable to vegetable
5、 oil methyl esters obtained from lauricoils, such as coconut oil and palm kernel oil.1.2 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.3 This standard does not purport to address all of thesafety concerns, if any, associated
6、 with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D4307 Practice for Preparation of Liquid Blends for Use asAnaly
7、tical StandardsE355 Practice for Gas Chromatography Terms and Rela-tionshipsE594 Practice for Testing Flame Ionization Detectors Usedin Gas or Supercritical Fluid Chromatography3. Terminology3.1 Definitions:3.1.1 biodiesel (B-100), nfuel comprised of mono-alkylesters of long chain fatty acids derive
8、d from vegetable oils oranimal fats.3.1.2 bonded glycerin, nglycerin portion of the mono-,di-, and triglyceride molecules.3.2 Definitions of Terms Specific to This Standard:3.2.1 total glycerin, nsum of free and bonded glycerin.3.3 This test method makes reference to many common gaschromatographic p
9、rocedures, terms, and relationships. Detaileddefinitions can be found in Practices E355 and E594.4. Summary of Test Method4.1 The sample is analyzed by gas chromatography, aftersilyating with N-methyl-N-trimethylsilyltrifluoracetamide(MSTFA). Calibration is achieved by the use of two internalstandar
10、ds and four reference materials. Mono-, di-, and trig-lyceride are determined by comparing to monoolein, diolein,and triolein standards respectively. Average conversion factorsare applied to mono-, di-, and triglycerides to calculate thebonded glycerin content of the sample.5. Significance and Use5.
11、1 Free and bonded glycerin content reflects the quality ofbiodiesel. A high content of free glycerin may cause problemsduring storage, or in the fuel system, due to separation of theglycerin. A high total glycerin content can lead to injectorfouling and may also contribute to the formation of deposi
12、ts atinjection nozzles, pistons, and valves.6. Apparatus6.1 Chromatographic SystemSee Practice E355 for spe-cific designations and definitions.6.1.1 Gas Chromatograph (GC)The system must be ca-pable of operating at the conditions given in Table 1.6.1.2 Column, open tubular column with a 5 % phenylpo
13、ly-dimethylsiloxane bonded and cross linked phase internal coat-ing. The column should have an upper temperature limit of atleast 400C. Columns, either 10 m or 15 m in length, with a0.32 mm internal diameter, and a 0.1 m film thickness havebeen found satisfactory. Any column with better or equivalen
14、t1This test method is under the jurisdiction of ASTM Committee D02 onPetroleum Products and Lubricants and is the direct responsibility of D02.04.0L onGas Chromatography Methods.Current edition approved Nov. 1, 2010. Published January 2011. Originallyapproved in 2000. Last previous edition approved
15、in 2010 as D658410. DOI:10.1520/D6584-10A.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1*A Summary of Chan
16、ges section appears at the end of this standard.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.chromatographic efficiency and selectivity can be used. It isrecommended thata2to5metre 0.53 mm high temperatureguard column be installed
17、from the injector to the analyticalcolumn. This allows the use of autoinjectors and also increasescolumn life.6.2 Electronic Data Acquisition System:6.2.1 Integrator or Computer, capable of providing realtime graphic and digital presentation of the chromatographicdata is recommended for use. Peak ar
18、eas and retention timesshall be measured by computer or electronic integration.6.2.2 This device must be capable of performing multilevelinternal-standard-type calibrations and be able to calculate thecorrelation coefficient (r2) and internal standard calculationsfor each data set.7. Reagents and Ma
19、terials7.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.3Other grades may b
20、e usedprovided it is first ascertained that the reagent is of sufficientpurity to permit its use without lessening the accuracy of thedetermination.7.2 n-Heptane, reagent grade.7.3 N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA),reagent grade.7.4 Pyridine, reagent grade.7.5 Carrier Gas, hydrogen
21、 or helium of high purity. Addi-tional purification is recommended by the use of molecularsieves or other suitable agents to remove water, oxygen, andhydrocarbons. Available pressure must be sufficient to ensure aconstant carrier gas flow rate.7.6 Microlitre Syringes, 100 L and 250 L capacity.7.7 Sc
22、rew Cap Vials, with polytetrafluoroethylene (PTFE)-faced septa, 10 mL capacity.8. Preparation of Apparatus8.1 Install and condition the column in accordance withmanufacturer or suppliers instructions. After conditioning,attach column outlet to flame ionization detector inlet andcheck for leaks throu
23、ghout the system. If leaks are found,tighten or replace fittings and recheck for leaks before proceed-ing.9. Calibration and Standardization9.1 Preparation of Calibration StandardsPrepare stan-dards using fresh compounds listed in Table 2 according toPractice D4307. Weigh the components directly int
24、o thevolumetric flasks specified and record the mass to the nearest0.1 mg. Dilute the volumetric flasks to mark with pyridine.Store the calibration standards in a refrigerator when not in use.9.2 Standard SolutionsPrepare the five standard solutionsin Table 3 by transferring the specified volumes by
25、 means ofmicrolitre syringes to 10 mL septa vials.Add to each of the fivestandard solutions 100 L of MSTFA. Close the vial and shake.Allow the vial to stand for 15 to 20 min at room temperature.Add approximately 8 mL n-Heptane to the vial and shake.9.3 Chromatographic AnalysisIf using an automatic s
26、am-pler, transfer an aliquot of the solution into a glass GC vial andseal with a TFE-fluorocarbonlined cap.9.4 StandardizationAnalyze the calibration standards un-der the same operating conditions as the sample solutions.Inject 1 L of the reaction mixture into the cool on-columninjection port and st
27、art the analysis. Obtain a chromatogramand peak integration report. For each reference substance,determine the response ratio (rspi) and amount ratio (amti) foreach component using Eq 1 and 2.rspi5 Ai/As! (1)where:Ai= area of reference substance, andAs= area of internal standard.amti5 Wi/Ws! (2)wher
28、e:Wi= mass of reference substance, andWs= mass of internal standard.9.4.1 Prepare a calibration curve for each reference compo-nent by plotting the response ratios (rspi), as the y-axis, versusthe amount ratios (amti), as the x-axis.3Reagent Chemicals, American Chemical Society Specifications, Ameri
29、canChemical Society, Washington, DC. For Suggestions on the testing of reagents notlisted by the American Chemical Society, see Annual Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC
30、), Rockville,MD.TABLE 1 Operating ConditionsInjectorCool on column injectionSample size 1 LColumn Temperature ProgramInitial temperature 50C hold 1 minRate 1 15C / min to 180CRate 2 7C / min to 230CRate 3 30C / min 380C hold 10 minDefectorType Flame ionizationTemperature 380CCarrier GasType Hydrogen
31、 or helium measured at 50CFlow rate 3 mL/minTABLE 2 Stock SolutionsCompound CAS No.ApproximateMass (mg)VolumetricFlask Size(mL)Glycerin 56-81-5 25 501-Mono cis-9-octadecenoyl-rac-glycerol (monoolein)111-03-5 50 101,3-Di cis-octadecenoylglycerol(diolein)2465-32-9 50 101,2,3-Tri cis-octadecenoylglycer
32、ol(triolein)122-32-7 50 10(S) - (-) -1,2,4-Butanetriol - (InternalStandard 1)42890-76-6 25 251,2,3-Tridecanolylglycerol (tricaprin) -(Internal Standard 2)621-71-6 80 10D6584 10a129.5 Calculate the correlation coefficient r2value for eachreference component in the calibration set using Eq 3. The r2va
33、lue should be at least 0.99 or greater. If the above criteria forr2are not met, rerun the calibration or check instrumentparameters and hardware.r25(xy!2(x2!(y2!(3)where:x 5 Xi2 x (4)y 5 Yi2 y (5)and:Xi= amtiratio data point,x = average values for all amtidata pointsYi= corresponding rspidata points
34、,y = average values for all rspidata points.9.6 Calibration FunctionsFor each reference calibrationfunctions are calculated in the form:AxAis5FaxSWxWisDG1 bx(6)where:Wx= mass of reference substance, mg,Wis= mass of internal standard, mg,Ax= peak area of reference substance,Ais= peak area of internal
35、 standard,ax= slope of the calibration function, andbx= intercept of the calibration function.10. Procedure10.1 Set the instrument operating variables to the valuesspecified in Table 1. Weigh to the nearest 0.1 mg approximately100 mg of sample directly into a 10 mL septa vial. Usingmicrolitre syring
36、es, add exactly 100 L of each internalstandard and MSTFA. Shake the vials, and allow to set for 15to 20 min at room temperature. Add approximately 8 mL ofn-Heptane to the vial and shake.10.2 Inject 1 L of the reaction mixture into the coolon-column injection port and start the analysis. Obtain achro
37、matogram and peak integration report.10.3 Peak IdentificationIdentify peaks by comparison ofretention times to the standards. For identification of additionalpeaks, use the relative retention times given in Table 4 and thereference chromatograms given in Fig. 1. Mono-, di-, andtriglycerides are sepa
38、rated according to carbon numbers (CN).10.4 Monoglyceride consists of the four overlapping peakswith relative retention times (RRT) of 0.76 and 0.83 to 0.86with respect to the internal standard tricaprin. A pair of peaks,methyl esters with a carbon number of 24, may appear withRRT of 0.80 to 0.82, a
39、nd should not be included in thecalculation of monoglyceride.10.5 Diglyceride is also primarily separated according tocarbon number, but due to varying double bonds in themolecules, baseline resolution of the peaks does not occur. Thegrouping of 3 to 4 peaks with RRT of 1.05 to 1.09 (CN 34, 36,and 3
40、8) shall be attributed to diglyceride. Carbon number alsoseparates triglyceride. Peaks with RRT of 1.16 to 1.31 (CN 52,54, 56, and 58) should be included in the calculation.11. Calculation and Report11.1 After identifying the peaks, measure the areas of thepeaks identified as glycerin, mono-, di-, a
41、nd triglyceride. Usingthe slope and y-intercept of the calibration functions, calculatethe mass of each as follows:11.1.1 Glycerin:G 5FWis1agGSFAgAisG bgDF100WG(7)where:G = mass percentage of glycerin in sample,Ag= peak area of glycerin,Ais1= peak area of Internal Standard 1,Wis1= weight of Internal
42、 Standard 1, mg,W = weight of sample, mg,ag= slope of the calibration function,bg= intercept of the calibration function.11.1.2 Individual Glyceride:Glj5FWis2aolGSFAgljAis2G bo1DF100WG(8)where:Glj= mass percentage of individual glyceride in sample,Aglj= peak area of individual glyceride,Ais2= peak a
43、rea of Internal Standard 2,Wis2= weight of Internal Standard 2, mg,W = weight of sample, mg,aol= slope of the calibration function for mono, di-, ortriolein, andbol= intercept of the calibration function for mono, di, ortriolein.11.1.3 Calculation of Total Glycerin:total glycerin 5 free glycerin 1 b
44、ound glycerin (9)where:free glycerin = glycerin determined in Eq 7,bound glycerin = ( (GlM,GlD,GlT)TABLE 3 Standard SolutionsStandard Solution Number 1 2 3 4 5L of glycerin stock solution 10 30 50 70 100L of monoolein stock solution 20 50 100 150 200L of diolein stock solution 10 20 40 70 100L of tr
45、iolein stock solution 10 20 40 70 100L of butanetriol stock solution 100 100 100 100 100L of tricaprin stock solution 100 100 100 100 100TABLE 4 Approximate Relative Retention TimesComponent Use InternalStandardRelative RetentionTimeGlycerin 1 0.851,2,4 Butanetriol 1.00Internal Standard 1Monopalmiti
46、n 2 0.76Monoolein, monolinolein 2 0.83-0.86monolinolenin, and monostearinTricaprin 1.00Internal Standard 2Diglycerides 2 1.05-1.09Triglycerides 2 1.16-1.31D6584 10a13where:GlM= 0.2591 3(monoglyceride, mass % determined inEq 8,GlD= 0.1488 3(diglyceride, mass % determined in Eq8, andGlT= 0.1044 3(trig
47、lyceride, mass % determined in Eq8.11.2 Report the total monoglyceride, total diglyceride, totaltriglyceride, and free and total glycerin to the nearest 0.001mass %.12. Precision and Bias12.1 The precision of this procedure, as determined bystatistical examination of the 2006 interlaboratory test re
48、sults,4obtained from 12 laboratories on 12 B-100 biodiesel samplesfrom a variety of sources, is as follows:12.1.1 RepeatabilityThe difference between successiveresults obtained by the same operator with the same apparatusunder constant operating conditions on identical test material,would in the lon
49、g run, in the normal and correct operation ofthe test method, exceed the following values in on case intwenty.12.1.1.1 Total Glycerin Repeatability:r 5 5.405E202* TG 1 0.5164! (10)TG = the calculated result for total glycerin expressed as amass % of the glycerin content in the sample, andr = repeatability.12.1.1.2 Free Glycerin Repeatability:r 5 2.339E202* FG 11.000E204!0.4888(11)FG = the calculated result for free glycerin expressed as amass % of the glycerin content in the sample, andr = repeatability.12.1.1.3 Total Mo
